Isolation and identification of pyogenic cocci

Experiment Three

Objective of Experiment To mTo masteraster basicbasic principleprinciple and and

methodmethod,which use to isolate and identify ,which use to isolate and identify pyogenic cocci pyogenic cocci from clinical from clinical sspecimens.pecimens.

To To diagnosediagnose clinical clinical disease and guide disease and guide doctors to select proper medicines .doctors to select proper medicines .

Specimens

(Gram-stain) Morphological characteristics (Microscopic examination)

Isolation culture

Smears

(blood agar plate)

Colonial characteristics

Hemolytic reaction

Pigmentation

PROCEDURE

morphology observation

colonial

typical colonies

stained and observe

Pure culture Antibiotic

susceptibility test

Direct identification

Identified method for Staphylococci

Gram-stainGram-stain Isolation Isolation and cultureand culture Pure culturePure culture

DirectDirect identifidentificationication

Staphylococci are Gram-positive cocci, typically arranged in clumps or Grape-like clusters

Direct identification

The mannitol fermentation test.The mannitol fermentation test. The Coagulase TestThe Coagulase Test The Dnase TestThe Dnase Test The phage typing test The phage typing test Animal ExperimentAnimal Experiment

The mannitol fermentation test.

Inoculate the bacteria into a mannitol Inoculate the bacteria into a mannitol micro-tube,incubate at 37micro-tube,incubate at 3700C for 18h.SC for 18h.S.aureus will ferment mannitol to prod.aureus will ferment mannitol to produce acid,which causes the medium tuce acid,which causes the medium to turn yellowo turn yellow. .

Test of Microbiological experiment While a patient is infected by pathogenic enterobacteria (While a patient is infected by pathogenic enterobacteria (Sal

monella),how to diagnose with microbiological methods in la,how to diagnose with microbiological methods in lab?b?

(simple procedure)(simple procedure)

While a patient is infected by pyogenic cocci,how to diagnosWhile a patient is infected by pyogenic cocci,how to diagnose with microbiological methods in lab?(simple procedure)e with microbiological methods in lab?(simple procedure)

While a patient is infected by influenza virus,how to diagnosWhile a patient is infected by influenza virus,how to diagnose with microbiological methods in lab?(simple procedure)e with microbiological methods in lab?(simple procedure)

The Coagulase Test

Coagulase is an enzyme converting fibrinogen inCoagulase is an enzyme converting fibrinogen into fibrin promoting blood clotting. to fibrin promoting blood clotting.

It might be a virulence factor with the coagulateIt might be a virulence factor with the coagulated blood around the bacteria protecting them frod blood around the bacteria protecting them from the immune system. m the immune system.

Coagulase-negative strains are often as pathogeCoagulase-negative strains are often as pathogenic as coagulase-positive strains.nic as coagulase-positive strains.

The Slide Coagulase Test

The Tube coagulase Test

Left tube coagulase positive Right tube coagulase negative

The Dnase Test Inoculate Dnase agar plates with a loop so Inoculate Dnase agar plates with a loop so

that the growth is in plaques about 1 cm in that the growth is in plaques about 1 cm in diameter.Incubate at 37diameter.Incubate at 3700C overnight.Flood C overnight.Flood the plate with 1 N hydrochloric acid.Clearithe plate with 1 N hydrochloric acid.Clearing around the colonies indicates Dnase acng around the colonies indicates Dnase activity.The hydrochloric acid reacts with untivity.The hydrochloric acid reacts with unchanged deoxyribonucleic acid to give a clchanged deoxyribonucleic acid to give a cloudy precipitate.A few other bacteria,e.g. oudy precipitate.A few other bacteria,e.g. Serratia,may give a positive reaction. Serratia,may give a positive reaction.

The phage typing test

This test is used to trace the infective This test is used to trace the infective agent in epidemiology if necessary.agent in epidemiology if necessary.

It is usually not done for routine cliniIt is usually not done for routine clinical purposecal purpose. .

Animal Experimentvomit excrement remaindered food

Isolation and identification of bacteria

Meat soup media

filter injection

6—8w cat

observationFood poisoning

The antibiotic susceptibility test This test is helpful for the treatment This test is helpful for the treatment

of S.aureus infectionof S.aureus infection.. materialsmaterials

StaphaureusStaphaureus （（ isolated from the pus of isolated from the pus of a patienta patient ）） . .

Several kinds of filter paper Several kinds of filter paper （（ each coneach contains different kinds of antibioticstains different kinds of antibiotics ））

Nutrient agar plateNutrient agar plate

The antibiotic susceptibility test

Streaking the staphaureus on agar plate

(thoroughly covered the plate)

Put 4 kinds of paper contained different antibiotics on the plate (each paper are far away about 2 cm)

Incubate at 370C for 18-24 hours.

Observe the results the plates The sensitivity of the organism is indicated by the diameter

of the zone of growth inhibition.

Laboratory Diagnosis of Pathogenic Enterobacterial Infection

Dimidiation of the enterobacteria according to the fermentation of lactose

Lactase fermenters:Lactase fermenters: saprophytic and commensalsaprophytic and commensal

Escherichia Klebsiella Citrobater Escherichia Klebsiella Citrobater

EnterobacterEnterobacter

Non lactase fermentersNon lactase fermenters: : pathogens pathogens

Salmonella Shigella some Citrobacter Salmonella Shigella some Citrobacter

Proteus SerratiaProteus Serratia

Colonial characteristic observation

Specimens isolation Gram Staining

(SS/EMB plate) Serological identification

TSI

Biochemical reaction

Procedure

Specimens

Different specimens should be taken depeDifferent specimens should be taken depending on the kind and the process of the nding on the kind and the process of the disease.disease.

blood blood

bone marrowbone marrow

Urine Urine

stoolstool

Isolation

Culture medium: S.S agar Culture medium: S.S agar Method: streak plateMethod: streak plate Result: Result:

Pathogenetic colonies: middle size, redPathogenetic colonies: middle size, red

Suspect colonies: colorless, small, opSuspect colonies: colorless, small, opaque aque

Biochemical reactions of Salmonella, etc

  

Species bottom slope H2S motilitySpecies bottom slope H2S motility E.coli AG AG - +E.coli AG AG - + Salmonella A - +/- + Salmonella A - +/- + Other Salmonella AG - +/- +Other Salmonella AG - +/- + Shigella A - +/- -Shigella A - +/- -

bottom: ferment dextrose slope: lactosebottom: ferment dextrose slope: lactose A:acid AG: acid and gasA:acid AG: acid and gas

Antigens of salmonella O antigen polysaccharide of LPS somatic antigen

used to divide Salmonella to 42 groups A-Z groups are pathogenic stable to heat (remains activity at 100 )℃H antigen flagella antigen divide Salmonella tow phase: special no special

Vi antigen related to the virulence of Salmonella sensitive to heat ( lose activity at 60 )℃

SalmonellaSalmonella

Serological Identification of Salmonella

Select the specimenSelect the specimen

React with A-Z polyvalent antiserumReact with A-Z polyvalent antiserum

React with several individual React with several individual types of anti-O and anti-H types of anti-O and anti-H

antiserum antiserum

React with anti-Vi React with anti-Vi antiserumantiserum

Agglutination reaction Agglutination reaction

(( ＋＋ ) () ( －－ ) )

Identify its group and phage

Gram StainGram Stain

Gram stain of

Nocardia asteroidesacid-fast stain of Nocardi

a asteroides

flagella